Elevated levels of UDP-Glucuronic acid mitigate drug resistance and stress response through a protease and a nucleotide sugar transporter in Cryptococcus gattii

UDP-glucuronic acid (UDP-GlcUA) is a nucleotide sugar that plays important roles in many organisms and excessive UDP-GlcUA in the cell causes many defects in the cellular processes. In Cryptococcus spp., mutations in the UXS1 gene which encodes an enzyme that converts UDP-GlcUA into UDP-xylose trigger high level accumulation of UDP-GlcUA and effectuate resistance to the antifungal drug 5-fluorocytosine. Here, we show that elevation of UDP-GlcUA affects several cellular processes including growth rate, ability to grow at various stress conditions, and resistance to fluorine containing analogs. RNA-seq analyses reveal that UXS1 deletion leads to the identification of three differentially expressed endopeptidase genes, notably PEP401. Lack of PEP401 from the uxs1 mutant background reduces the UDP-GlcUA levels and reverts all the phenotypes of the uxs1 mutant toward the wild-type characteristics. Particularly, high levels of UDP-GlcUA not only regulate expression of PEP401 at RNA and protein levels, but it also enhances the proteolytic activity of total protein extracts in a PEP401-dependent manner, establishing a functional link between nucleotide sugar metabolism and proteolytic regulation. Moreover, the UDP-GlcUA transporter gene, UUT1, can further modulate the levels of UDP-GlcUA in the uxs1 pep401 double mutant and manifests the drug resistance phenotypes observed in the uxs1 mutant. Taken together, these findings reveal a previously unrecognized regulatory network that connects UDP-GlcUA metabolism to protease-mediated cellular processes and to the transportation of UDP-GlcUA. This newly identified interplay provides a foundation for targeting nucleotide sugar metabolism and protease regulation in the development of improved therapeutic strategies against cryptococcosis. WM276 = WT strain C2053 = UXS1 deletion mutant C2104 = UXS1 deletion mutant with UGD1 (G19A) C2061 = UGD1 (G19A) strain Total RNAs were isolated by the Trizol reagent (Invitrogen, Carlsbad, CA) and RNAeasy mini kit (Qiagen). RNAs were treated with Turbo RNase-free DNase (Ambion) to remove genomic DNA. polyA-RNA was purified from total RNA, processed, and paired-end directional libraries (150 bp) were prepared and sequenced using an Illumina HiSeq 4000 platform (Novogene, Davis, CA).